Small RNA sequencing using next generation sequencing technologies (sRNA-seq) is invaluable for small RNA profiling and discovery in fields such as cancer, stem cell biology, and epigenetic gene regulation. sRNA-seq library preparation has historically suffered from three major drawbacks; severe bias, the need for gel-based purification, and the lack of low-input protocols. Reducing the formation of adapter-dimer products is a key aspect in the successful creation of these libraries.
The need to purify final small RNA sequencing libraries by gel, typically by PAGE gel, is due to the small difference in the size of adapter-dimer molecules versus insert-containing molecules following the PCR step of library preparation. In typical DNA or RNA library prep, insert-containing molecules are at least 100 bp larger than adapter-dimer molecules, and thus can be removed using Solid Phase Reversible Immobilization (SPRI) magnetic beads. However, since insert-containing molecules are only ˜20 bp larger than adapter-dimer molecules in small RNA libraries, SPRI size selection is not feasible, and gel-based selection must be performed. The need for gel-based size selection greatly limits both throughput and automation potential of small RNA library preparation, as only a limited number of libraries can be run on a single gel and it is a labor-intensive process that is not amenable to automation.
The lack of low-input protocols for sRNA-seq is also related to adapter-dimer formation. Small RNA sequencing is somewhat unique in that additional PCR cycles result in negligible bias; thus it should theoretically be possible to create low-input small RNA libraries by using a high number of PCR cycles. However, adapter-dimer present in the libraries will also be greatly amplified, which eventually leads to a library where adapter-dimer products are extremely abundant, making it difficult to isolate insert-containing products and leading to sequencing data where very few of the reads are useful. A number of methods have been developed to reduce adapter-dimer formation in small RNA library preparation, but unfortunately none are effective at reducing adapter-dimer formation to such an extent that gel-free or low-input small RNA library preparation is possible
There are currently multiple methods for reduction of adapter-dimer products. In one of these methods, a complementary oligonucleotide is annealed to the 3′ adapter following the first ligation step, which converts excess 3′ adapter from single-stranded DNA to double-stranded DNA. The double-stranded DNA is a poor substrate for the T4 RNA ligase 1 enzyme used in the subsequent reaction, resulting in reduced formation of adapter-dimer products.
Traditional methods of construction of sRNA libraries have been shown to suffer from severe bias, resulting in final sequencing results that do not accurately represent relative abundances of small RNAs in the starting material. This bias can be greatly reduced through the use of oligonucleotide adapters with randomized bases at the ligation junctions. However, the strategy of hybridization of a complementary oligonucleotide does not work well to reduce adapter-dimer formation when using adapters with randomized ends. Thus, purification of intermediary ligation products by polyacrylamide gel electrophoresis (PAGE) was often used in sRNA library preparation protocols utilizing adapters with randomized ends. Purification of products by PAGE has many disadvantages, so a gel-free adapter-dimer reduction strategy was developed. This strategy requires the use of a proprietary reagent combined with isopropanol and SPRI beads to deplete excess 3′ adapter following the first ligation step, thus reducing formation of adapter-dimer in the final library. However, it should be noted that neither this strategy nor strategies using conventional (non-randomized end containing) adapters are typically effective at reducing adapter-dimer formation to such a level that final purification by PAGE can be replaced by a SPRI-based method.
Thus, there is a need for methods for reducing the formation of adapter-dimer products in certain molecular biology techniques, including creating nucleic acid sequencing libraries. For example but not limited to, RNA libraries for use in next generation sequencing of RNA, including small RNA.